Chem 135 Exam 2 Notes v2.txt

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  1. Peptidyl proryl isomerase (PPI)
    Catalyzes cis/trans isomerization of peptide bonds to pro
  2. Protein disulfide isomerase (PDI)
    Catalyzes disulfide exchange (forms correct set of disulfide bonds
  3. Molecular chaperones and chaperonins
    Prevent contact bewtween proteins to prevent precipitation
  4. Chaperons
    • HSP70/HSP40, HSP90 (heat shock proteins, eurkaryote)
    • DnaK/DnaJ (prokaryote)
    • Bind to and release unfolded proteins in an ATP-dependent manner
    • Cover up apolar surfaces while bound
  5. Chaperonins
    • HSP60 (barrel)/HSP10(cap) - euk
    • GroEl/GroES - pro (essential for E. coli growth)
    • Consist of two heptameric "barrels" and a heptameric "cap"
    • Isolates unfolded protein and allows it to fold by binding to apolar regions and enclosing in isolated space
  6. Myoglobin (Mb)
    • Higher affinity for O2
    • Reservoir for O2 in marine mammals
    • Facilitates O2 transport in rapidly respiring tissues
    • Eliminates nitric oxide by converting to NO3-
  7. Hemoglobin (Hb)
    • Carries O2 from lungs to tissue
    • Binding O2 instead of H2O requires FE(II) oxidation state
  8. Fractional Saturation (Y)
    Fraction of binding sites with ligand (e.g. O2)
  9. The ___ of Fe(II) to Fe(III) occurs in vivo which is ___. Two enzymes have evolved to deal with this:___ and ___; ____.
    oxidation, bad. MetMb and MetHb, reductases
  10. lung_P_O2 = ___. tissue_P_O2 = ___. For Mb, P_50 = ___. => Mb is ___.
    100 Torr. 30 Torr. 2.84 Torr. saturated in lung and tissues
  11. Hemoglobin O2 binding - Sigmoidally =>
  12. Cooperative binding
    Binding of first ligand affects the affinity with which the next ligand is bound
  13. Equation for infinite coopertivity by Hill
    see notes
  14. Hill coefficient
    For hemoglobin, about 3
  15. n_h is an indicator of cooperativity
    • > 1 : possitive coop
    • = 1 : no coop
    • < 1 : negative coop
  16. The closer n_h is to n, the ___ the extent of cooperativity.
  17. Hill plot
  18. H_b / O2 affinity ___ with decreasing pH.
  19. CO2/bicarbonate equilibrium
    CO2 + H2O <--> H2CO3 <--> HCO3- + H+
  20. Oxy conformation, __ form, is __ binding.
    R, tightly
  21. Deoxy conformation, __ form, is __ binding.
    T, weakly
  22. R form is ___ acidic than T form.
  23. ___ acidity further releases O2.
  24. 2,3-BPG binds to and ___ the ___ (or ___) conformation of Hb, thus ___ Hb/O2 affinity.
    stabilizes, T, deoxy, lowering
  25. Chloride ___ Hb/O2 affinity by participating in a ___ in the ___ (but not ___) conformation.
    diminishes, salt bridge, T, R
  26. The iron ion stays within the Heme plane in ___ conformation, __ affinity.
    oxy, increasing
  27. in HbF (fetal), 143 serine (instead of histidine) sidechain resides at 2,3-BPG binding site, ___ affinity for 2,3-BPG, thus ___ overall O2 affinity.
    lowering, increasing
  28. Myoglobin is a ___ that has 153aa's most of which are in ___ alpha helices.
    monomer, eight
  29. In myoglobin, what draws Fe(II) out of heme plane?
    Histidine, F8 (8th aa in 6th alpha helix)
  30. In oxy myoglobin, the conformation of oxy(R) and deoxy(T) are ___.
    essentially identical, i.e. not much change in binding O2
  31. Hemoglobin is a 65kD ___ in which the structures of the R and T states are ___.
    alpha2 beta2 tetramer, substantially different
  32. Normaly Hb P50 is about ___ torr; less than ___ of O2 in lungs is released to tissue, ___ torr.
    26, half, 30
  33. Discuss hemoglobin structure.
    • Approximately spheroid: 65x55x50 Angstroms
    • 4 subunits: alpha2, beta2 tetramer
    • 65 kD
    • Tertiary structures of alpha, beta, and Mb are NEARLY identical, but only 18% homology and no D-helix in alpha => lots of diff sequences can fold into same/similar patterns
    • Intersubunit interactions are between alpha-beta, not a-a or b-b
    • Conformations between deoxy(T) and oxy(R) are VERY DIFFERENT
    • Tighter contact betw ai-bi => rearangement at ai-bj
  34. Give an explanation for the "Bohr" effect.
    • T state stabilized by 8 salt bridges all of which break during T to R change
    • pKa increases in oxy state for NH3+
    • Also, CO2 + H20 <-> H2CO3 <-> HCO3- + H+
  35. Substrate
    Ligand of focus (e.g. O2 for Hb)
  36. Effector
    Ligand that alters binding affinity
  37. Homotropic effects
    Effector is identical to substrate
  38. Heterotropic effects
    Effector is different than substrate
  39. What kind of effect is binding for O2?
    Positive homotrophic effect
  40. What kind of effect is binding for 2,3-BGP?
    Negative heterotropic effect
  41. What are the postulates for the MWC (symmetric/concerted) cooperativity model?
    • Ro <=> To, L = [To]/[Ro]
    • An allosteric protein consists of a set of functionally identical subunits
    • Each subunit can exist in two (or more) conformations
    • The ligand can bind to either conformation, but with different affinity
    • The conformation change is ALL or NONE (i.e. concerted)
  42. Discuss the theoretical curves for Hb and the actual
    • Hb: sigmoidal (between theoretical)
    • R-only: All at once (left of Hb) - similar to end of Hb curve
    • T-only: slow (right of Hb) - similar to beginning of Hb curve
  43. Summarize the MWC coop model.
    Binding affinity depends ONLY on the conformational state of the protein
  44. What are the postulates for the KNF (sequential/induced fit) cooperativity model?
    • Ligand binding to a subunit changes conformation of that subunit
    • Cooperative effects arise as consequence of change in conformation on neighboring subunits
    • Affinity depends on number of ligands bound
  45. Define enzyme
    Biological catalyst than can produce very large rate increases under mild (physiological) conditions.
  46. A catalyst must ___ the magnitude of ___ by either ___ or ___.
    decrease, deltaG^dagger, lowering dagger (T.S.), raising S_bar
  47. Describe the M&M equation.
    v(rate) = v_max[S]/(k_m + [S])
  48. Describe the relationship between v_max, k_cat, and [Eo].
    v_max = k_cat * [Eo]
  49. What is the steady state assumption?
    [ES] is constant over time
  50. Describe k_cat.
    • Turnover number
    • k_cat = v_max/[Eo]
    • Maximum number of S converted to P per unit time by a molecule of enzyme
    • Ranges from lysozyme (0.5 molecules/sec) to catalase (10^8 molecules/sec)
  51. Describe Km.
    • Apparent dissociation constant, i.e. measure of enzymes affinity for a substrate
    • Km = [S]_0.5, i.e. [S] when v is half maximal (i.e. v_max/2)
    • Lower Km => higher affinity for S
  52. Describe k_cat/Km.
    • A measure of SPECIFICITY for a substrate
    • Given two enzymes with equal concentration, the enzyme will catalyze the substrate with the larger ratio preferentially
    • Also a measure of enzyme efficiency (some enzymes approach theoretical maximum)
  53. Describe the linearization of the M&M equation.
    • Take the inverse and plot as a line
    • 1/v = (Km/v_max)*(1/[S]) + 1/v_max
  54. Four enzymes approaching "perfection"
    • 1. Acetylcholine esterase: neurotransmitter
    • 2. Carbonic anhydrase: Convert CO2 to water-soluble form
    • 3. Triose phosphate isomerase: provides quick energy
    • 4. Beta-lactamase: developed by bacteria to survive antibiotics like penicillin
  55. What are two properties of reversible inhibition of enzyme activity?
    • Involves non-covalent binding
    • Always some enzyme not bound
  56. What are 4 types of reversible inhibition?
    • Competitive
    • Uncompetitive
    • Mixed
    • Non-competitive
  57. Describe competitve inhibition.
    • Inhibitor resembles substrate, i.e. S and I compete for binding to E.
    • App_v_max = v_max, i.e. more substrate is needed to "drown out" inhibitor.
    • Doubling [I] => doubling of slope (app_v_max constant, app_Km increases)
  58. Describe uncompetitive inhibition.
    • I binds ONLY to ES complex
    • Both app_v_max and app_Km decrease => increase in affinity for S
    • As long as any I is present, you will get some ESI => less product
    • As I increases, graph shifts to "left" since app_v_max and app_Km decrease.
  59. Describe mixed inhibition.
    • I binds to BOTH E and ES with different affinities.
    • app_v_max decreases.
    • app_Km = (alpha/alpha')Km
    • If alpha > alpha' (i.e. if I binds to E with higher affinity than to ES) => app_Km increases
  60. Describe non-competitive inhibition.
    • I binds to BOTH E and ES with SAME affinity
    • app_v_max decreases and app_Km remains the same => change in slope; same x-int
  61. Summarize app_v_max, app_Km, and app_(V/K)
    • Competitive: V(V/K)/alphaalpha*K
    • Uncompetitive:V/alpha'(V/K)K/alpha'
    • Mixed:V/alpha'(V/K)/alpha(alpha'/alpha)*K
    • Non-compet:V/alpha'(V/K)/alphaK
  62. Summarize app_v_max, app_Km, and app_(V/K)
    • Competitive: V (V/K)/alpha alpha*K
    • Uncompetitive: V/alpha' (V/K) K/alpha'
    • Mixed: V/alpha' (V/K)/alpha (alpha'/alpha)*K
    • Non-compet: V/alpha' (V/K)/alpha K
  63. Describe irreversible inhibition
    • Knocks out enzyme (on physiological timescale)
    • Active site directed reagents
    • Mechanism based
  64. Describe active site directed reagents for irreversible inhibition
    Binds to active site of enzyme and modifies residue at that site
  65. Describe mechanism based irreversible inhibition
    Inhibitor, for example, makes a covalent bond with enzyme so that it no longer functions
  66. List the catalytic mechanisms
    • General acid/base
    • Electrostatic catalysis
    • Proximity and oritentation effects
    • Preferential binding of the transition state
    • Covalent catalysis
  67. Describe general acid/base mechanism
    • H+ transfer is part of the rate determining step (whereas in specific, it's not)
    • e.g. RNase A
    • Does not change "mode" of the reaction
  68. Describe electrostatic catalysis
    • Fully developed charges/dipoles at active site favorably interact with developing charges/dipoles
    • e.g. Carbonic anhydrase
    • Does not change "mode" of the reaction
  69. Describe proximity and oritentation effects
    • Decreases deltaG_dagger of the reaction to an unspecified extent
    • Depends on inter vs. intra molecular interactions and conformations
    • Does not change "mode" of the reaction
  70. Describe preferential binding of the transition state
    • Binds T.S. with higher affinity that S resulting in a decrease in deltaG_dagger
    • Does not change "mode" of the reaction
  71. Describe Covalent catalysis
    • Enzyme provides a new pathway by which to convert substrates to product that involves the formation of a covalently bound intermediate
    • e.g. Type I aldoase
  72. Coenzyme
    • Non-protein chemical compound that is loosely-bound to an enzyme and is required for its activity.
    • Pyridoxal phosphate (PLP)
  73. Serine proteases
    • Large class of proteins that catalyze peptide bond hydrolysis via an acyl enzyme intermediate
    • Great deal of homology => divergent evolution
    • Each contains an essential catalytic triad: ser, his, asp
  74. Chymotripsin
    • H57, D102, S195
    • At least 4 different versions evolved separately
    • Reactive nucleophile is a serine sidechain
  75. Experimental evidence for Essential Triad
    • S195: knockout => no rxn; inactivated by DIPF unless S is present
    • H57: pH in basic form matches H57; TPCK inactivates enzyme and labels H57
    • D102: D102N mutant of enzyme has a k_cat value 10^4 times smaller than that of wildtype
  76. Experimental evidence for acyl enzyme intermediate
    • Burst kinetics: (at least) two steps, 2nd of which is rate-determining
    • Common hydrolysis rates: Different indvidual rates, but same rate in presense of enzyme (rate-determining)
    • Same partitioning ratios: same ratios => common acyl intermediate
  77. Describe lysozyme
    • Isolated from hen egg white
    • 14.7 kD consisting of 129 aa's that catalyzes the hydrolysis of the glycosidic linkage between NAM and NAG in bacterial cell walls
    • "cleans up" bacteria (not anti-bacterial)
    • Ellipsoid, 30x30x45A with deep cleft in one face
    • Cleft has six binding sites for monosaccharides, A-F
    • Cleavage site: D-E
  78. Essential residues of lysozyme
    • D52: polar environment (pKa 3.5)
    • E35: hydrophobic pocket (pKa 6.5)
    • Bell-shaped ph/rate profile
    • Alkylation of ionized carboxyl groups inactivate enzyme; presence of substrate protecting alkylation preserves active enzyme => D52 in active site
    • An E35Q mutant binds substrate with higher affinity, but shows less than 1% of activity of wildtype
  79. Mechanism for lysozyme
    • Double displacement/inversion: covalently-bound intermediate found in 1990
    • Retention of configuration by double-inversion
    • NOT Sn1 as previously thought (with blocking enzyme preserving configuration)
  80. Relative rate between NAG4 and NAG5 has a large jump indicating ___.
    that it MUST (not just MIGHT) span the cleavage site
  81. List the regulatory mechanisms of enzyme activity.
    • Alteration of enzyme concentration usually by altering transcription rate of gene (slow)
    • Limited proteolysis (slow)
    • Interacting with regulatory proteins (fast); responses to organismal needs
    • Covalent modification (fast); e.g. ATP phosphorylation; responses to organismal needs
    • Allosteric effects; solute binds far from active site but changes conformation; fast; response to cellular need
  82. Example of regulatory mechanisms: Epinephrin on glucose metabolism
    • Interaction with regulatory proteins (x3)
    • Covalent interaction (x2)
    • Several amplifications of signal
  83. Example of allosteric effect
    • ATCase: catalyzes the committed step in prokaryotic pyrimidine (U, C) nucleotide biosynthesis
    • CTP and UTP are inhibitors of enzyme activity (negative feedback)
    • ATP is an activator of enzyme activity
  84. Describe rate/[asp] curve with ATCase
    • Sigmoidal
    • Inhibitor: right-shifts curve (L=1250)
    • Activator: left-shifts curve (L=75)
  85. In terms of R_o and T_o, a large L implies ___.
    preferential binding to low-affinity form (T)
  86. Why does ATP activate ATCase?
    It's a purine nucleotide, and equilibrium wants equal rates of synthesis of purines and pyrimidines
  87. What is PALA?
    a bisubstrate analog that binds tightly to ATCase
  88. ATCase operates according to the ___ model of cooperativity.
    MWC (p.567-8)
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Chem 135 Exam 2 Notes v2.txt
Chem135 Biochem exam2
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